Proteins and nucleic acids employ only a small fraction of the available functionality. In vitro molecular evolution efforts include diversification of a starting molecule into related variants from which desired molecules are chosen. Methods used to generate diversity in nucleic acid and protein libraries include whole genome mutagenesis (Hart et al., Amer. Chem. Soc. (1999), 121: 9887-9888), random cassette mutagenesis (Reidhaar-Olson et al,. Meth. Enzymol. (1991), 208: 564-86), error-prone PCR (Caldwell, et al. (1992), PCR Methods Applic. (1992), 2: 28-33), and DNA shuffling using homologous recombination (Stemmer (1994) Nature (1994), 370: 389-391). After diversification, molecules with novel or enhanced properties can be selected.
Methods that enable recombination to take place at defined sites without sequence homology have been recently described. For example, it is possible to recombine unrelated protein-encoding genes by using synthetic oligonucleotides to encode each desired crossover (O'Maille (2002) J. Mol. Biol. 321: 677-91; and Tsuji (2001) Nuc. Acids Res. 29:E97). Although this strategy can result in a high likelihood of preserving function after diversification, many fewer sites of recombination, and therefore, fewer novel structures are accessible than if crossover sites are randomly generated. Alternatively, methods allowing a single nonhomologous crossover of two protein-encoding genes have been developed (Sieber (2001) Nat. Biotechnol. 19: 456-60; and Ostermeier (1999) Nat. Biotechnol 17: 1205-9), and additional nonhomologous recombination events can be obtained by fragmenting and homologously recombining the resulting genes (Lutz (2001) Proc. Natl. Acad. Sci. USA 98: 11248-5317). Despite efforts to enhance the number of crossovers obtained, existing methods for diversifying proteins by nonhomologous recombination have thus far yielded only modest numbers of recombination events (three or fewer per 500 base pair (bp) in protein-encoding sequences, with even fewer crossovers (one to two per 500 bp) among sequences encoding active proteins (Kawarasaki (2003) Nuc. Acids Res. 31: e12618).
Accordingly a need exists for a simple, effective method of diversifying nucleic acids proteins.